The focus of this application is to establish the relationship between structure and function of the pre-mRNA branch site, the RNA formation that contains the attacking nucleophile for the first of pre-mRNA splicing. The long-range objective of our work is to understand how RNA and protein components of the eukaryotic splicing apparatus assemble, define splice sites, and catalyze the removal of noncoding sequences (introns) from pre-mRNA molecules. Splicing is an integral step in the maturation of eukaryotic RNAs, and alternative splice site selection is a source of developmental stage- and tissue-specific protein diversity. Since errors in splicing are the basis of certain cancers and neurodegenerative diseases, an understanding of the splicing process is of biomedical importance. The spliceosome of eukaryotic nuclei is a dynamic assembly of five recyclable small nuclear (sn)RNAs and numerous proteins, whereas the Group II intron, found in certain prokaryotes and eukaryotic organelles, is a single RNA molecule comprising six secondary structural domains. The two splicing systems remove introns via the identical chemical mechanism and stereochemistry, and share certain sequence similarities, but only the Group II intron can carry out the splicing reaction in vitro in the absence of proteins. It is therefore likely that the two systems share evolutionary ancestry and that RNA also plays a major catalytic role in the spliceosomal reaction. Gaps in structural information have limited comparison. The Specific Aims are: 1) determine the contributions of individual RNA-RNA interactions to the spliceosomal branch site structure and their implications on splicing activity; and 2) probe the structure of the pre-mRNA branch site of the Group II self-splicing intron in order to compare it with its spliceosomal counterpart. Structural studies in solution will be carried out by homonuclear and heteronuclear NMR; the effect of various mutations and modifications on splicing activity will be assessed by perturbations to product formation of in vitro splicing assays. A number of innovative NMR techniques will be exploited to assist in determination of high resolution solution structural models and orientation of RNA domains. The proposed experiments will provide new insights into the ancestry and the molecular basis of splicing activity in eukaryotic cells.